Ambion, Inc. proposes to develop methods and reagents that will make it possible to compare the expression profiles of small RNA samples (<1 microgram of RNA) using microarray analysis. The key element of the proposal is an amplification scheme that will allow two RNA populations to be co-verse transcribed and co-amplified in a single tube using the same reagents. Simultaneous amplification will obviate the effects of tube-to-tube variation that plagues current amplification schemes. Following amplification, sequences derived from the two input RNA samples will be labeled with different fluorescent dyes and the two target populations will be hybridized to a single microarray. In addition to a method for RNA amplification, Ambion proposes to improve the sensitivity of microarrays by developing an enhanced microarray hybridization solution by systematically assessing the benefits of known hybridization enhancers. Finally, an RNA reference population will be developed that will provide a standard against which data from many different experiments can be compared. PROPOSED COMMERCIAL APPLICATIONS: The proposed research has many commercial applications. Success in Phase I will result in an enhanced microarray hybridization system and internal control problems that will be useful for all microarray expression research. Phase II success will result in an amplification system that can be applied by individual researchers using limited RNA samples. Ambion will use the amplification scheme to provide nucleic acids from difficult to obtain samples to the research community.